This program is extended from our continuing efforts to investigate the immune response to the hepatitis C virus (HCV) in both humans and experimental animals. In extensive earlier studies, we identified immunodominant and neutralizing epitopes on the hepatitis C virus that will now be further investigated to examine the relationship between immune response and persistent infection. In FY 95 and FY 96, we initiated a new project to examine the potential of nucleic acid vaccination for the prevention and/or treatment of HCV infection. The long-term goal of both the basic immunology studies and the DNA vaccine studies is to develop models for immune therapy of chronic viral infections of the liver. One of the advantages of genetic immunization is that the endogenously expressed proteins can be recognized by class I MHC molecules and expressed on the cell surface. The MHC-antigen complex on the cell surface can be recognized by cytotoxic T lymphocytes (CTL) which, in turn, are activated and attack infected cells. The possibility of inducing an immune response to HCV core protein using DNA immunization provides an attractive alternative to classic vaccination. There are many problematic issues related to the vaccine development for hepatitis C. One major concern is the genetic instability of the infectious agent. There are two hypervariable regions in the putative HCV envelope proteins. Immune escape mutants have been attributed to mutations in these regions. Experimentally infected chimpanzees and HCV-infected patients have been found to repeat bouts of infection with either homologous or new strains of HCV. This failure to develop protective immunity links to the high chronicity rate in HCV infection. Directly inducing strong cell-mediated immunity, especially protective cytotoxic T lymphocyte responses, may not only help in preventing initial HCV infection, but may serve as a mechanism for immune modulation to overcome existing infection. Using the mouse model, we were able to evaluate the induction of antibodies to several different plasmid constructs containing both HCV structural and non-structural genes. We were also able to develop assays to measure both humoral and cell-mediated immune responses, including CTL activities, in the mouse model. In the past year, we have tested the genetic sequences of many HCV-related immunogens to establish the best candidate DNA vaccine. We have also studied methods of vaccine delivery and immunity augmentation procedures; accumulated extensive experience in measuring humoral and cell-mediated immunity; and developed effective immunization strategies in small experimental animals. We believe we are now ready to test our findings in the only animal model susceptible to HCV infection, the chimpanzee. Protocols are being written for DNA vaccination in the chimpanzee using constructs containing genes for HCV core and envelope proteins.